The homeostasis of life is maintained by a network composed of numerous proteins. Since various diseases can be perceived as a failure of homeostasis, the amount of each protein present in a living body can be used as a biomarker for elucidation of the molecular mechanism or diagnosis of diseases, or as a criterion for therapeutic prognosis. Although both high comprehensiveness and high sensitivity are required in protein detection and quantification to achieve this purpose, there is presently no technique that is sufficient for practical use.
Examples of known high-sensitivity protein detection methods include immunochemical methods, such as western blotting utilizing antibodies specific to individual proteins. However, in immunochemical methods, the acquisition of specific antibodies is a prerequisite, and the obtained results greatly depend on the quality of the antibody used. Furthermore, in order to detect plural proteins by an immunochemical method, all antibodies against individual proteins must be prepared, and analysis must be repeated using each individual antibody. However, making such comprehensive analysis is substantially impossible.
Examples of known methods for comprehensive analysis of proteins include proteome analysis methods using a mass spectrometer as basic technology. However, according to conventional proteome analysis methods, detecting trace proteins is extremely difficult. For example, two-dimensional electrophoresis, which is a typical proteomic expression analysis method, can only detect proteins with high expression levels. Even with the use of quantitative shotgun proteomics, which is a combination of LC-MS/MS with stable isotope labeling (SILAC, ICAT, iTRAQ), the number of proteins that can be detected is only several hundreds to about 3,000. When the number of detections is within such a range, detecting or quantifying trace proteins is impossible (see Non-patent Literature (NPL) 1 to 3). Furthermore, the purpose of these methods is generally relative quantification, rather than absolute quantification, of proteins. It is thus difficult to derive a quantitative relationship between proteins by comparing quantitative values of the proteins individually obtained in different research and testing laboratories by using such methods.
As a method for overcoming the problem of the conventional protein quantification analysis, multiple reaction monitoring (MRM) (also referred to as “selective reaction monitoring” (SRM)), which has been used for quantitative analysis of low molecular compounds, has been proposed to be used for peptide quantification (see Non-patent Literature (NPL) 4). However, obtaining MS/MS spectral information on the target peptide beforehand is necessary to perform MRM.
In general, the number of peptides generated from one protein by enzymatic digestion, etc., may range from several tens to several hundreds. Selection of the target peptide to be subjected to MRM is a very important step in specifying the sensitivity of this method. At present, the selection of the target peptide for MRM is made by utilizing a measured spectrum obtained by shotgun proteome analysis; or depends on a method of theoretical estimation under specific conditions.
However, because the actual spectrum data of trace proteins is rarely obtained, and theoretical estimation does not always guarantee the selection of highly sensitive peptides, the development of a method of efficiently selecting an MRM target peptide is necessary to perform comprehensive high-sensitivity MRM.
Thus, the MRM method is currently recognized only as a methodology for reviewing and confirming the results obtained by shotgun proteome analysis, etc., and is not expected to be used as a large-scale screening method.